Information on EC 2.1.1.235 - dTDP-3-amino-3,6-dideoxy-alpha-D-glucopyranose N,N-dimethyltransferase

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The expected taxonomic range for this enzyme is: Streptomyces fradiae

EC NUMBER
COMMENTARY hide
2.1.1.235
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RECOMMENDED NAME
GeneOntology No.
dTDP-3-amino-3,6-dideoxy-alpha-D-glucopyranose N,N-dimethyltransferase
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
2 S-adenosyl-L-methionine + dTDP-3-amino-3,6-dideoxy-alpha-D-glucopyranose = 2 S-adenosyl-L-homocysteine + dTDP-3-dimethylamino-3,6-dideoxy-alpha-D-glucopyranose
show the reaction diagram
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Biosynthesis of antibiotics
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dTDP-alpha-D-mycaminose biosynthesis
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Polyketide sugar unit biosynthesis
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SYSTEMATIC NAME
IUBMB Comments
S-adenosyl-L-methionine:dTDP-3-amino-3,6-dideoxy-alpha-D-glucopyranose 3-N,N-dimethyltransferase
The enzyme is involved in the biosynthesis of mycaminose, an essential structural component of the macrolide antibiotic tylosin, which is produced by the bacterium Streptomyces fradiae.
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
physiological function
additional information
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structure model of TylM1 with bound S-adenosyl-L-methionine and dTDP-phenol: Model of the Michaelis complex in stereo. The C-3' amino group is positioned to attack the methyl group of S-adenoyl-L-methionine. dTDP-mycaminose binding pocket structure, overview
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
2 S-adenosyl-L-methionine + dTDP-3-amino-3,4,6-trideoxy-alpha-xylo-hexopyranose
2 S-adenosyl-L-homocysteine + dTDP-3-dimethylamino-3,4,6-trideoxy-alpha-xylo-hexopyranose
show the reaction diagram
kcat/KM is about 3fold lower than kcat/Km for the natural substrated TDP-3-amino-3,6-dideoxy-alpha-D-glucopyranose
i.e. TDP-alpha-D-desosamine
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?
2 S-adenosyl-L-methionine + dTDP-3-amino-3,6-dideoxy-alpha-D-glucopyranose
2 S-adenosyl-L-homocysteine + dTDP-3-dimethylamino-3,6-dideoxy-alpha-D-glucopyranose
show the reaction diagram
S-adenosyl-L-methionine + dTDP-3-methylamino-3,6-dideoxy-alpha-D-glucopyranose
S-adenosyl-L-homocysteine + dTDP-3-dimethylamino-3,6-dideoxy-alpha-D-glucopyranose
show the reaction diagram
chemically prepared monomethylated compound is a substrate for TylM1 and could be swiftly converted by TylM1 to the dimethylated product. TylM1 catalyzes an N,N-dimethylation reaction by way of a monomethylated intermediate. Since the monomethylated amino group is intrinsically a better nucleophile than the unsubstituted amino group, the reaction rate of this SN2-type methyl transfer reaction is expected to be higher for the second half reaction than for the first methylation reaction
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?
additional information
?
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NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
2 S-adenosyl-L-methionine + dTDP-3-amino-3,6-dideoxy-alpha-D-glucopyranose
2 S-adenosyl-L-homocysteine + dTDP-3-dimethylamino-3,6-dideoxy-alpha-D-glucopyranose
show the reaction diagram
additional information
?
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the enzyme also catalyzes the methylation of dTDP-3-amino-3,6-dideoxy-alpha-D-galactopyranose, EC 2.1.1.236
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
S-adenosyl-L-methionine
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
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TylM1 is not a Mg2+-dependent methyltransferase
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.045 - 0.93
dTDP-3-amino-3,6-dideoxy-alpha-D-glucopyranose
0.0468
dTDP-3-methylamino-3,6-dideoxy-alpha-D-glucopyranose
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pH 7.5, 24°C
0.0989 - 0.117
S-adenosyl-L-methionine
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0126 - 0.75
dTDP-3-amino-3,6-dideoxy-alpha-D-glucopyranose
0.54
dTDP-3-methylamino-3,6-dideoxy-alpha-D-glucopyranose
Streptomyces fradiae
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pH 7.5, 24°C
0.09 - 0.86
S-adenosyl-L-methionine
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0135 - 3.8
dTDP-3-amino-3,6-dideoxy-alpha-D-glucopyranose
4448
11.5
dTDP-3-methylamino-3,6-dideoxy-alpha-D-glucopyranose
Streptomyces fradiae
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pH 7.5, 24°C
41647
0.91 - 7.35
S-adenosyl-L-methionine
24
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.5
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assay at
8.5
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assay at
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
27000
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2 * 27000, SDS-PAGE
27427
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2 * 27427, calculated from sequence
27530
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x * 27530, calculated from sequence
55200
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gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
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x * 27530, calculated from sequence
homodimer
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2 * 27000, SDS-PAGE; 2 * 27427, calculated from sequence
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
enzyme in complex with S-adenosyl-L-homocysteine and dTDP-3-N-methylamino-3,6-dideoxyglucose., X-ray diffraction structure determination and analysis at 1.6 A resolution
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hanging drop method of vapor diffusion method, high resolution X-ray structures of TylM1, one in which the enzyme contains bound SAM and dTDP-phenol and the second in which the protein is complexed with S-adenosyl-L-homocysteine and dTDP-3-amino-3,6-dideoxyglucose, its natural substrate. Combined, these two structures, solved to 1.35 A and 1.79 A resolution, respectively, show the orientations of SAM and the dTDP-linked sugar substrate within the active site region. Specifically, the C-30 amino group of the hexose is in the correct position for an in-line attack at the reactive methyl group of S-adenosyl-L-methionine. High-resolution X-ray models show that the observed perturbations in the kinetic constants of the mutant enzynes H123A and H123N are not due to major changes in their threedimensional folds. Most likely the proton on the C-3' amino group is transferred to one of the water molecules lining the active site pocket as catalysis proceeds
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GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
concentrated TylM1 protein is stable and can endure repeated freeze/thaw cycles without losing noticeable activity
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inclusion of 0.1 mM S-adenosyl-L-methioninet in buffers is crucial to prevent TylM1 from precipitating during the purification
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TylM1 is unstable at low concentrations, requiring the addition of bovine serum albumin (1 mg/mL) to the assay buffer during kinetic studies of the
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-80 °C concentrated TylM1 protein is stable for at least 2 years
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
inclusion of 0.1 mM AdoMet in buffers is crucial to prevent TylM1 from precipitating during the purification
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expression in Escherichia coli
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expression in Escherichia coli. Heterologous expression of the tylM1 gene in the desVI deletion mutant of Streptomyces venezuelae. TylM1 is a competent substitute for DesVI
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
H123A
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mutant protein retains catalytic activity, the kcat/Km is reduced to 1.8% compared to the wild-type enzyme
H123N
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mutant protein retains catalytic activity, the kcat/Km is reduced to 0.37% compared to the wild-type enzyme
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
medicine
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the enzyme is involved in biosynthesis of D-mycaminose. D-Mycaminose is an unusual dideoxy sugar found attached to the antibiotic tylosin, a commonly used veterinarian therapeutic